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Immune Complexes Induce TNF-Α and BAFF Production from U937 Cells by HMGB1 and RAGE Tory Mediators8,9

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Immune Complexes Induce TNF-Α and BAFF Production from U937 Cells by HMGB1 and RAGE Tory Mediators8,9 European Review for Medical and Pharmacological Sciences 2017; 21: 1810-1819 Immune complexes induce TNF-a and BAFF production from U937 cells by HMGB1 and RAGE X.-J. GAO1,2, Y.-Y. QU2, X.-W. LIU2, M. ZHU2, C.-Y. MA2, Y.-L. JIAO2, B. CUI2, Z.-J. CHEN3, Y.-R. ZHAO2 1Center of Laboratory Medicine, The Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai, China 2Central Laboratory, Provincial Hospital Affiliated to Shandong University, Jinan, China 3Research Center for Reproductive Medicine, Provincial Hospital Affiliated to Shandong University, Jinan, China Abstract. – OBJECTIVE: This study investi- Key Words: gated the effects of immune complexes (ICs) on Immune complexes, U937 cells, High mobility group tumor necrosis factor α (TNF-α) and B cell-ac- box protein 1, Receptor for advanced glycation end tivating factor (BAFF) production from U937 products, Systemic lupus erythematosus. cells and further explored the mechanism. MATERIALS AND METHODS: U937 cells were incubated with necrosis supernatant or system- ic lupus erythematosus (SLE) sera alone, or Introduction their combination. The expression of TNF-α and BAFF was determined by Real-time poly- Systemic lupus erythematosus (SLE) is a mul- merase chain reaction and enzyme-linked im- ti-systemic involvement and chronic progressive munosorbent assay. High mobility group box protein 1(HMGB1) A-box was produced by gene autoimmune disorder characterized by production recombination. HMGB1 A-box and anti-receptor of autoantibodies against nucleic components such for advanced glycation end products (RAGE) as DNA and nucleosomes and formation of immu- antibody were adopted in the blocking experi- ne complexes (ICs) due to polyclonal B cell acti- ments. The importance of DNA for cytokine in- vation1,2. Being inactive in its free form, however, duction was investigated by DNase treatment. mammalian DNA in the form of ICs has potent RESULTS: The combination of necrosis su- immunostimulatory properties3,4. The necessity for pernatant and SLE sera induced the expres- sion of TNF-α and BAFF significantly increased complex formation suggests that endogenous DNA compared to necrosis supernatant or SLE se- must exist in association with another moiety or ra alone. Recombinant HMGB1 A-box protein structure to enable access to the corresponding re- was purified, and TNF-α and BAFF production, ceptor. High mobility group box protein 1 (HMGB1) which were induced by this combination, was is known as a non-histone nuclear protein that binds blocked via HMGB1 A-box and anti-RAGE anti- DNA in a non-sequence-specific manner, modi- body. Moreover, we found that DNA component fying chromosomal architecture and facilitating is important for the immunostimulatory activity 5 of this combination. gene transcription . HMGB1 has been highlighted CONCLUSIONS: ICs containing DNA can pro- as a pro-inflammatory mediator when it is released mote TNF-α and BAFF production in U937 cells, from cells. Binding of HMGB1 to the receptor for and this process can be mediated by HMGB1 advanced glycation end products (RAGE), Toll-like and RAGE. One possible mechanism of increas- receptor-2 (TLR-2) or TLR-4 can lead to the release ing BAFF production in SLE is proposed in this of pro-inflammatory cytokines6,7. However, highly study whereby B cell activation, antibody pro- purified HMGB1 has a weak or no pro-inflamma- duction and ICs stimulated monocytes may cre- ate a vicious cycle that leads to B cell hyper- tory activity; for full activity, it may need to form a activity, which can be of importance for SLE complex with another component to activate inflam- etiopathogenesis. mation by enhancing effects of other pro-inflamma- 1810 Corresponding Author: Yueran Zhao, MD; e-mail: [email protected] Immune complexes induce TNF-α and BAFF production from U937 cells by HMGB1 and RAGE tory mediators8,9. Of note, HMGB1 can bind avidly demonstrated that ICs containing DNA could pro- to immunostimulatory molecules such as lipopoly- mote the increase of TNF-α and BAFF production saccharide (LPS), DNA or interleukin (IL)-1β so as in U937 cells via HMGB1 and RAGE. Therefore, to promote their activity in a synergistic fashion9,10. the presented data may help to better understand the Given that increased DNA or nucleosomes in the cytokine disturbance and pathogenesis of SLE. blood of SLE has been attributed to secondary ne- crosis, it appears reasonable to expect that released nucleosomes or DNA may carry along the tightly Materials and Methods bound pro-inflammatory HMGB1. Urbonaviciute et al11 demonstrated that nucleosomes were complexes Culture of U937 Cells and Induction with HMGB1 in the supernatants of secondary ne- of Cell Necrosis crotic cells and HMGB1 was present in circulating The human monocyte line U937 (ATCC 1593) ICs from SLE patients. Monocyte is a key compo- was cultured in Modified Roswell Park Memorial nent of the innate immune system involved in the Institute (RPMI)-1640 medium (TransGen Biote- regulation of adaptive immune responses. Aberrant ch, Beijing, China) supplemented with 10% fetal activation of the adaptive immune system in SLE calf serum (FCS) (HyClone, Logan, UT, USA), is augmented through the dysregulated production L-glutamine (2 mM, MP Biomedicals, Shanghai, of cytokines1. Abnormally activated monocytes/ China), HEPES (20 mM, MP Biomedicals, Shan- macrophages are major sources of some cytokines ghai, China), penicillin (60 μg/ml, Solarbio, Bei- and are usually found in the sera and tissues from jing, China), and streptomycin (100 μg/ml, So- 12-14 patients with SLE , which may promote the acti- larbio, Beijing, China), at 37ºC in 5% CO2. The vation of auto-reactive T and B cells. Among the- induction of U937 cell necrosis was performed by se cytokines, tumor necrosis factor-alpha (TNF-α) repeat freeze thawing as previously described14. contributes to tissue inflammation and damage15, Cell debris was removed by centrifugation (400 and B cell can activate factor from the TNF family g for 10 min, GTR22-1, Beijing Era Beili Cen- (BAFF) that promotes the survival and proliferation trifuge Co., Ltd, Beijing, China) and necrotic cell of B lymphocytes7,16. BAFF, also known as B lym- supernatant was isolated and stored in -80°C for phocyte stimulator (BLyS), is mainly produced by subsequent preparation of ICs. monocytes. Circulating BAFF levels are elevated and correlate positively with anti-dsDNA antibody Serum Preparation titers and disease activity in SLE patients17,18. Huma- SLE sera were prepared from the blood samples nized anti-BAFF antibody and BAFF decoy recep- of SLE patients in our hospital who fulfilled the tor significantly reduced the level of autoantibodies American College of Rheumatology (ACR) 1997 and B-cell activation in SLE19. Thus, BAFF is likely revised criteria for the classification of SLE23. Ve- to play a critical role in the activation of antigen-dri- nous blood was collected from SLE patients during ven autoimmune B cells in human SLE. In SLE, one a flare or with active disease based on clinical clas- explanation for excessive cytokine production is the sification criteria into tubes without anticoagulants. presence of nucleic acid-containing ICs in the serum After isolated, sera were screened by anti-dsDNA of lupus patients. Recent reports13,20,21 have shown antibody ELISA test (Euroimmun, Lübeck, Ger- that nucleic acid-containing ICs are capable of in- many). Only sera with positive anti-dsDNA titer ducing TNF or IFN-α production from peripheral greater than 1:100, Shanghai KaLang Biologi- blood mononuclear cells (PBMCs), plasmacytoid cal Technology (Co., Ltd, Shanghai, China) were dendritic cells (PDCs) and B cells. Moreover, the in- selected and mixed. The mixed sera were passed teraction between HMGB1 and RAGE triggers the through a 0.45 μm filter and stored at -80°C. Also, activation of PDCs and B cells in response to lupus sera from healthy blood donors were prepared and ICs22. Given these findings, we suggest that ICs play used in some control experiments. All the samples their biologic roles primarily through stimulating were collected after informed written consent was monocyte activation. With many monocytic cha- obtained from all participants, and this study was racteristics, U937 cells may be chosen as a model to approved by Ethics Committee of Shandong Uni- investigate the effect of itself or environmental agen- versity. ts on monocytic function. The aim of this study was to investigate the effects of ICs on TNF-α and BAFF Preparation of Immune Cells (ICs) production from U937 cells and further explored Necrotic U937 cell supernatant and mixed SLE the mechanism for these effects. The results herein sera obtained above were used for preparation 1811 X.-J. Gao, Y.-Y. Qu, X.-W. Liu, M. Zhu, C.-Y. Ma, Y.-L. Jiao, B. Cui, Z.-J. Chen, Y.-R. Zhao of ICs. ICs were prepared according to previous TNF-a and BAFF mRNA Assays reports13,24. In this study, ICs were preformed by Total RNA was extracted using the TRIzol re- combining 0.5% (vol/vol) SLE sera and 12.5% agent (Invitrogen, Carlsbad, CA, USA). Then, 1 (vol/vol) necrosis supernatant in the complete μg of total RNA from each sample was used as a medium according to our preliminary experimen- template in the reverse transcription reaction to ts. The control groups were designed as follows: synthesize cDNA with random hexamer primers 12.5% necrosis supernatant or 0.5% SLE sera alo- according to Fermentas RT kit (Fermentas, Glen ne, the combination of 12.5% necrosis supernatant Burnie, MD, USA). All samples were reverse tran- and 0.5% normal sera, or 0.5% normal sera alone. scribed under the same condition (42ºC for 60 min) We found that the combination of 12.5% necrosis and from the same reverse transcription master mix supernatant and 0.5% normal sera did not modify to minimize differences in reverse transcription ef- the effect of necrosis supernatant alone, and 0.5% ficiency.
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